Elased Khalid M, Cool David R, Morris Mariana
Department of Pharmacology and Toxicology, Wright State University School of Medicine, Dayton, OH 45435, USA.
Hypertension. 2005 Oct;46(4):953-9. doi: 10.1161/01.HYP.0000174601.30793.b1. Epub 2005 Aug 15.
This article demonstrates the applicability of quantitative proteomics to assays of proteolytic enzyme activity. A novel assay was developed for measurement of renin and angiotensin-converting enzyme (ACE) activity in plasma. The method was validated in animal models associated with alterations of the renin angiotensin system (RAS). Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) with a ProteinChip Array technology, plasma renin and ACE1 could be measured in <0.5 microL of plasma. Plasma is incubated with peptide substrates for renin and ACE, tetradecapeptide (TDP), and angiotensin I (Ang I), respectively. The reactions mixtures are spotted onto the ProteinChip WCX2 and detected using SELDI-TOF-MS. Peak height or area under curve for TDP, Ang I, and angiotensin II (Ang II) peaks are measured. There was a linear relationship between disappearance of substrate and appearance of products for both renin and ACE (R2=0.95 to 0.98). ACE1 activity was blocked with chelating agents (EDTA and 1,10 phenanthrolene), indicating action of a metalloprotease. The ACE1 inhibitor, captopril, selectively blocked ACE1. Renin activity was specifically blocked with renin inhibitor and was not affected by phenanthrolene or captopril. Animal models tested were Ang AT1a receptor-deficient and streptozotocin (STZ) diabetic mice. Plasma renin activity was increased >2-fold in AT1a(-/-) as compared with AT1a(+/+). In STZ diabetic mice, ACE1 was increased 2-fold as compared with controls. The advantage of the method is that it is tagless, does not require additional purification steps, and is extremely sensitive. The approach can be multiplexed and used for identification of novel substrates/inhibitors of the RAS.
本文展示了定量蛋白质组学在蛋白水解酶活性检测中的适用性。开发了一种用于测量血浆中肾素和血管紧张素转换酶（ACE）活性的新检测方法。该方法在与肾素血管紧张素系统（RAS）改变相关的动物模型中得到了验证。使用表面增强激光解吸/电离飞行时间质谱（SELDI-TOF-MS）和蛋白质芯片阵列技术，可在<0.5微升血浆中测量血浆肾素和ACE1。分别将血浆与肾素和ACE的肽底物、十四肽（TDP）和血管紧张素I（Ang I）一起孵育。将反应混合物点样到蛋白质芯片WCX2上，并用SELDI-TOF-MS进行检测。测量TDP、Ang I和血管紧张素II（Ang II）峰的峰高或曲线下面积。肾素和ACE的底物消失与产物出现之间均存在线性关系（R2 = 0.95至0.98）。ACE1活性被螯合剂（EDTA和1,10-菲咯啉）阻断，表明是金属蛋白酶的作用。ACE1抑制剂卡托普利可选择性阻断ACE1。肾素活性被肾素抑制剂特异性阻断，不受菲咯啉或卡托普利影响。所测试的动物模型为Ang AT1a受体缺陷型和链脲佐菌素（STZ）糖尿病小鼠。与AT1a(+/+)相比，AT1a(-/-)血浆肾素活性增加了2倍以上。在STZ糖尿病小鼠中，ACE1比对照组增加了2倍。该方法的优点是无标记、无需额外的纯化步骤且极其灵敏。该方法可进行多重分析，并用于鉴定RAS的新型底物/抑制剂。
